Distinct Effects of Moxifloxacin and Bedaquiline on Growing and 'Non-Culturable' Mycobacterium abscessus.

Mycobacterium abscessus has recently emerged as the cause of an increasing number of human infections worldwide. Unfortunately, it is highly resistant to existing drugs, and new specific agents to combat M. abscessus have not yet been found. The discovery of antibiotics that are effective not only against replicating but also against dormant and often recalcitrant cells is a daunting challenge. In this study, we developed a model of non-replicating M. abscessus, which represents a valuable screening tool for antibacterial agents. Thus, we demonstrated that, under a deficiency of potassium ions in the growth media and prolonged incubation, M. abscessus entered a 'non-culturable' state with a significant loss of colony-forming ability, but it retained viability, as confirmed using the most-probable-number (MPN) assay. The 'non-culturable' mycobacteria possessed decelerated cellular metabolism and noticeable differences in cell morphology from actively growing mycobacteria. 'Non-culturable' cells were used in a comprehensive screening of the efficacy of antibiotics, along with actively growing cells. Both CFU and MPN tests confirmed the prominent bactericidal effect of moxifloxacin on actively growing and 'non-culturable' M. abscessus, as proven by less than 0.01% of cells surviving after antibiotic treatment and prolonged storage. Bedaquiline exhibited a comparable bactericidal effect only on metabolically inactive non-culturable cells aged for 44 days. There were reductions ranging from 1000 to 10,000-fold in CFU and MPN, but it was not so efficient with respect to active cells, resulting in a bacteriostatic effect. The demonstrated specificity of bedaquiline in relation to inert non-replicating M. abscessus offers a new and unexpected result. Based on the findings of this research, moxifloxacin and bedaquiline can be regarded as potential treatments for infections caused by M. abscessus. In addition, a key outcome is the proposal to include the combination of viability assays for comprehensive testing of drug candidates. Relying on CFU-based assays alone resulted in overestimates of antibacterial efficacy, as demonstrated in our experiments.

Small RNA MTS1338 Configures a Stress Resistance Signature in Mycobacterium tuberculosis.

In the course of evolution, Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, has developed sophisticated strategies to evade host immune response, including the synthesis of small non-coding RNAs (sRNAs), which regulate post-transcriptional pathways involved in the stress adaptation of mycobacteria. sRNA MTS1338 is upregulated in Mtb during its infection of cultured macrophages and in the model of chronic tuberculosis, suggesting involvement in host-pathogen interactions. Here, we analyzed the role of MTS1338 in the Mtb response to macrophage-like stresses in vitro. The Mtb strain overexpressing MTS1338 demonstrated enhanced survival ability under low pH, nitrosative, and oxidative stress conditions simulating the antimicrobial environment inside macrophages. Transcriptomic analysis revealed that in MTS1338-overexpressing Mtb, the stress factors led to the activation of a number of transcriptional regulators, toxin-antitoxin modules, and stress chaperones, about half of which coincided with the genes induced in Mtb phagocytosed by macrophages. We determined the MTS1338 "core regulon", consisting of 11 genes that were activated in all conditions under MTS1338 overexpression. Our findings indicate that MTS1338 is a stress-induced sRNA that promotes Mtb survival in macrophages by triggering adaptive transcriptional mechanisms in response to host antimicrobial defense reactions.

Mycobacterium tuberculosis Infection: Control and Treatment.

Tuberculosis (TB) is the second leading infectious killer after COVID-19, causing 10 million new cases and claiming the lives of more than 1 [...].

Mycobacterium tuberculosis Dormancy: How to Fight a Hidden Danger.

Both latent and active TB infections are caused by a heterogeneous population of mycobacteria, which includes actively replicating and dormant bacilli in different proportions. Dormancy substantially affects M. tuberculosis drug tolerance and TB clinical management due to a significant decrease in the metabolic activity of bacilli, which leads to the complexity of both the diagnosis and the eradication of bacilli. Most diagnostic approaches to latent infection deal with a subpopulation of active M. tuberculosis, underestimating the contribution of dormant bacilli and leading to limited success in the fight against latent TB. Moreover, active TB appears not only as a primary form of infection but can also develop from latent TB, when resuscitation from dormancy is followed by bacterial multiplication, leading to disease progression. To win against latent infection, the identification of the Achilles' heel of dormant M. tuberculosis is urgently needed. Regulatory mechanisms and metabolic adaptation to growth arrest should be studied using in vitro and in vivo models that adequately imitate latent TB infection in macroorganisms. Understanding the mechanisms underlying M. tuberculosis dormancy and resuscitation may provide clues to help control latent infection, reduce disease severity in patients, and prevent pathogen transmission in the population.

A New Benzothiazolthiazolidine Derivative, 11726172, Is Active In Vitro, In Vivo, and against Nonreplicating Cells of Mycobacterium tuberculosis.

Tuberculosis (TB) still poses a global menace as one of the deadliest infectious diseases. A quarter of the human population is indeed latently infected with Mycobacterium tuberculosis. People with latent infection have a 5 to 10% lifetime risk of becoming ill with TB, representing a reservoir for TB active infection. This is a worrisome problem to overcome in the case of relapse; unfortunately, few drugs are effective against nonreplicating M. tuberculosis cells. Novel strategies to combat TB, including its latent form, are urgently needed. In response to the lack of new effective drugs and after screening about 500 original chemical molecules, we selected a compound, 11726172, that is endowed with potent antitubercular activity against M. tuberculosis both in vitro and in vivo and importantly also against dormant nonculturable bacilli. We also investigated the mechanism of action of 11726172 by applying a multidisciplinary approach, including transcriptomic, labeled metabolomic, biochemical, and microbiological procedures. Our results represent an important step forward in the development of a new antitubercular compound with a novel mechanism of action active against latent bacilli. IMPORTANCE The discontinuation of TB services due to COVID-19 causes concern about a future resurgence of TB, also considering that latent infection affects a high number of people worldwide. To combat this situation, the identification of antitubercular compounds targeting Mycobacterium tuberculosis through novel mechanisms of action is necessary. These compounds should be active against not only replicating bacteria cells but also nonreplicating cells to limit the reservoir of latently infected people on which the bacterium can rely to spread after reactivation.

Design of Anti-infectious Agents from Lawsone in a Three-Component Reaction with Aldehydes and Isocyanides.

The first effective synthetic approach to naphthofuroquinones via a reaction involving lawsone, various aldehydes, and three isocyanides under microwave irradiation afforded derivatives in moderate to good yields. In addition, for less-reactive aldehydes, two naphtho-enaminodione quinones were obtained for the first time, as result of condensation between lawsone and isocyanides. X-ray structure determination for 9 and 2D-NMR spectra of 28 confirmed the obtained structures. All compounds were evaluated for their anti-infectious activities against Plasmodium falciparum, Leishmania donovani, and Mycobacterium tuberculosis. Among the naphthofuroquinone series, 17 exhibited comparatively the best activity against P. falciparum (IC50 = 2.5 µM) and M. tuberculosis (MIC = 9 µM) with better (P. falciparum) or equivalent (M. tuberculosis) values to already-known naphthofuroquinone compounds. Among the two naphtho-enaminodione quinones, 28 exhibited a moderate activity against P. falciparum with a good selectivity index (SI > 36) while also a very high potency against L. donovani (IC50 = 3.5 µM and SI > 28), rendering it very competitive to the reference drug miltefosine. All compounds were studied through molecular modeling on their potential targets for P. falciparum, Pfbc1, and PfDHODH, where 17 showed the most favorable interactions.

Targeting Non-Replicating Mycobacterium tuberculosis and Latent Infection: Alternatives and Perspectives (Mini-Review).

Latent tuberculosis infection (LTBI) represents a major challenge to curing TB disease. Current guidelines for LTBI management include only three older drugs and their combinations-isoniazid and rifamycins (rifampicin and rifapentine). These available control strategies have little impact on latent TB elimination, and new specific therapeutics are urgently needed. In the present mini-review, we highlight some of the alternatives that may potentially be included in LTBI treatment recommendations and a list of early-stage prospective small molecules that act on drug targets specific for Mycobacterium tuberculosis latency.

Small RNA F6 Provides Mycobacterium smegmatis Entry into Dormancy.

Regulatory small non-coding RNAs play a significant role in bacterial adaptation to changing environmental conditions. Various stresses such as hypoxia and nutrient starvation cause a reduction in the metabolic activity of Mycobacterium smegmatis, leading to entry into dormancy. We investigated the functional role of F6, a small RNA of M. smegmatis, and constructed an F6 deletion strain of M. smegmatis. Using the RNA-seq approach, we demonstrated that gene expression changes that accompany F6 deletion contributed to bacterial resistance against oxidative stress. We also found that F6 directly interacted with 5'-UTR of MSMEG_4640 mRNA encoding RpfE2, a resuscitation-promoting factor, which led to the downregulation of RpfE2 expression. The F6 deletion strain was characterized by the reduced ability to enter into dormancy (non-culturability) in the potassium deficiency model compared to the wild-type strain, indicating that F6 significantly contributes to bacterial adaptation to non-optimal growth conditions.

Small Noncoding RNAs and Their Role in the Pathogenesis of Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis possesses a significant arsenal of strategies to combat immune defense of the host organism. Small noncoding RNAs, which constitute the largest group of regulatory RNAs, play an important role in the host-pathogen interactions and represent one of the levels of the regulation of interactions of microbial cells with their environment. The regulatory role of small RNAs in pathogenic bacteria is essential when rapid adaptation to the changing environmental conditions with further synchronization of metabolic reactions are required to ensure microbial survival and infection progression. During the past few years, eight small RNAs from M. tuberculosis have been functionally characterized, and targets for four of them have been identified. Small RNAs from M. tuberculosis and other pathogenic microorganisms were found to be one of the most important functional factors in the adaptive response to changing environmental conditions.

Mycobacterium tuberculosis Small RNA MTS1338 Confers Pathogenic Properties to Non-Pathogenic Mycobacterium smegmatis.

Small non-coding RNAs play a key role in bacterial adaptation to various stresses. Mycobacterium tuberculosis small RNA MTS1338 is upregulated during mycobacteria infection of macrophages, suggesting its involvement in the interaction of the pathogen with the host. In this study, we explored the functional effects of MTS1338 by expressing it in non-pathogenic Mycobacterium smegmatis that lacks the MTS1338 gene. The results indicated that MTS1338 slowed the growth of the recombinant mycobacteria in culture and increased their survival in RAW 264.7 macrophages, where the MTS1338-expressing strain significantly (p < 0.05) reduced the number of mature phagolysosomes and changed the production of cytokines IL-1ß, IL-6, IL-10, IL-12, TGF-ß, and TNF-α compared to those of the control strain. Proteomic and secretomic profiling of recombinant and control strains revealed differential expression of proteins involved in the synthesis of main cell wall components and in the regulation of iron metabolism (ESX-3 secretion system) and response to hypoxia (furA, whiB4, phoP). These effects of MTS1338 expression are characteristic for M. tuberculosis during infection, suggesting that in pathogenic mycobacteria MTS1338 plays the role of a virulence factor supporting the residence of M. tuberculosis in the host.

Design and Synthesis of Pyrano[3,2-b]indolones Showing Antimycobacterial Activity.

Latent Mycobacterium tuberculosis infection presents one of the largest challenges for tuberculosis control and novel antimycobacterial drug development. A series of pyrano[3,2-b]indolone-based compounds was designed and synthesized via an original eight-step scheme. The synthesized compounds were evaluated for their in vitro activity against M. tuberculosis strains H37Rv and streptomycin-starved 18b (SS18b), representing models for replicating and nonreplicating mycobacteria, respectively. Compound 10a exhibited good activity with MIC99 values of 0.3 and 0.4 µg/mL against H37Rv and SS18b, respectively, as well as low toxicity, acceptable intracellular activity, and satisfactory metabolic stability and was selected as the lead compound for further studies. An analysis of 10a-resistant M. bovis mutants disclosed a cross-resistance with pretomanid and altered relative amounts of different forms of cofactor F420 in these strains. Complementation experiments showed that F420-dependent glucose-6-phosphate dehydrogenase and the synthesis of mature F420 were important for 10a activity. Overall these studies revealed 10a to be a prodrug that is activated by an unknown F420-dependent enzyme in mycobacteria.

New Insights into the Mechanism of Action of the Thienopyrimidine Antitubercular Prodrug TP053.

The thienopyrimidine TP053 is an antitubercular prodrug active against both replicating and nonreplicating Mycobacterium tuberculosis (M. tuberculosis) cells, which requires activation by the mycothiol-dependent nitroreductase Mrx2. The investigation of the mechanism of action of TP053 revealed that Mrx2 releases nitric oxide from this drug both in the enzyme assays with purified Mrx2 and in mycobacterial cultures, which can explain its activity against nonreplicating bacilli, similar to pretomanid activated by the nitroreductase Ddn. In addition, we identified a highly reactive metabolite, 2-(4-mercapto-6-(methylamino)-2-phenylpyrimidin-5-yl)ethan-1-ol, which can contribute to the antimycobacterial effects on replicating cells as well as on nonreplicating cells. In summary, we explain the mechanism of action of TP053 on both replicating and nonreplicating M. tuberculosis and report a novel activity for Mrx2, which in addition to Ddn, represents another example of nitroreductase releasing nitric oxide from its substrate. These findings are particularly relevant in the context of drugs targeting nonreplicating M. tuberculosis, which is shown to be killed by increased levels of nitric oxide.

MTS1338, A Small Mycobacterium tuberculosis RNA, Regulates Transcriptional Shifts Consistent With Bacterial Adaptation for Entering Into Dormancy and Survival Within Host Macrophages.

Small non-coding RNAs play a significant role in bacterial adaptation to changing environmental conditions. We investigated the dynamics of expression of MTS1338, a small non-coding RNA of Mycobacterium tuberculosis, in the mouse model in vivo, regulation of its expression in the infected macrophages, and the consequences of its overexpression in bacterial cultures. Here we demonstrate that MTS1338 significantly contributes to host-pathogen interactions. Activation of the host immune system triggered NO-inducible up-regulation of MTS1338 in macrophage-engulfed mycobacteria. Constitutive overexpression of MTS1338 in cultured mycobacteria improved their survival in vitro under low pH conditions. MTS1338 up-regulation launched a spectrum of shifts in the transcriptome profile similar to those reported for M. tuberculosis adaptation to hostile intra-macrophage environment. Using the RNA-seq approach, we demonstrate that gene expression changes accompanying MTS1338 overexpression indicate reduction in translational activity and bacterial growth. These changes indicate mycobacteria entering the dormant state. Taken together, our results suggest a direct involvement of this sRNA in the interplay between mycobacteria and the host immune system during infectious process.

A New Antisense Phosphoryl Guanidine Oligo-2'-O-Methylribonucleotide Penetrates Into Intracellular Mycobacteria and Suppresses Target Gene Expression.

The worldwide spread of multidrug-resistant Mycobacterium tuberculosis strains prompted the development of new strategies to combat tuberculosis, one of which is antisense therapy based on targeting bacterial mRNA by oligonucleotide derivatives. However, the main limitation of antisense antibacterials is poor cellular uptake because of electrostatic charge. Phosphoryl guanidine oligo-2'-O-methylribonucleotides (2'-OMe PGOs) are a novel type of uncharged RNA analogues with high RNA affinity, which penetrate through the bacterial cell wall more efficiently. In this study, we investigated the uptake and biological effects of 2'-OMe PGO in mycobacteria. The results indicated that 2'-OMe PGO specific for the alanine dehydrogenase-encoding ald gene inhibited the growth of Mycobacterium smegmatis and downregulated ald expression at both the transcriptional and translational levels through an RNase H-independent mechanism, showing higher biological activity than its phosphorothioate oligonucleotide counterpart. Confocal microscopy revealed that the anti-ald 2'-OMe PGO was taken up by intracellular mycobacteria residing in RAW 264.7 macrophages without exerting toxic effects on eukaryotic cells, indicating that 2'-OMe PGO was able to efficiently cross two cellular membranes. In addition, 2'-OMe PGO inhibited the transcription of the target ald gene in M. smegmatis-infected macrophages. Thus, we demonstrated, for the first time, a possibility of targeting gene expression and inhibiting growth of intracellular mycobacteria by antisense oligonucleotide derivatives. Strong antisense activity and efficient uptake of the new RNA analogue, 2'-OMe PGO, by intracellular microorganisms revealed here may promote the development of novel therapeutic strategies to treat TB and prevent the emergence of drug-resistant mycobacterial strains.

Resuscitation of Dormant "Non-culturable" Mycobacterium tuberculosis Is Characterized by Immediate Transcriptional Burst.

Under unfavorable conditions such as host immune responses and environmental stresses, human pathogen Mycobacterium tuberculosis may acquire the dormancy phenotype characterized by "non-culturability" and a substantial decrease of metabolic activity and global transcription rates. Here, we found that the transition of M. tuberculosis from the dormant "non-culturable" (NC) cells to fully replicating population in vitro occurred not earlier than 7 days after the start of the resuscitation process, with predominant resuscitation over this time interval evidenced by shortening apparent generation time up to 2.8 h at the beginning of resuscitation. The early resuscitation phase was characterized by constant, albeit low, incorporation of radioactive uracil, indicating de novo transcription immediately after the removal of the stress factor, which resulted in significant changes of the M. tuberculosis transcriptional profile already after the first 24 h of resuscitation. This early response included transcriptional upregulation of genes encoding enzymes of fatty acid synthase system type I (FASI) and type II (FASII) responsible for fatty acid/mycolic acid biosynthesis, and regulatory genes, including whiB6 encoding a redox-sensing transcription factor. The second resuscitation phase took place 4 days after the resuscitation onset, i.e., still before the start of active cell division, and included activation of central metabolism genes encoding NADH dehydrogenases, ATP-synthases, and ribosomal proteins. Our results demonstrate, for the first time, that the resuscitation of dormant NC M. tuberculosis is characterized by immediate activation of de novo transcription followed by the upregulation of genes controlling key metabolic pathways and then, cell multiplication.

A rapid method for estimation of the efficacy of potential antimicrobials in humans and animals by agar diffusion assay.

Drug resistance continues to challenge traditional antimicrobial drugs and limit their clinical utility. This requires us to continue our search for new drug candidates with novel mechanisms of action against infectious diseases. We now describe a simple agar diffusion assay, which can be used as a general method for the rapid detection of antimicrobial activity of drug candidates in animal or human blood plasma for the ultimate prediction of the efficacy of potential drugs prior to clinical trials. We present an example for a clinical candidate against Mycobacterium tuberculosis.

Copper-related toxicity in replicating and dormant Mycobacterium tuberculosis caused by 1-hydroxy-5-R-pyridine-2(1H)-thiones.

With the emerging primary resistance of Mycobacterium tuberculosis to current drugs and wide distribution of latent tuberculosis infection, the need for new compounds with a novel mode of action is growing. Copper-mediated innate immunity and its antibacterial toxicity pose novel strategies for tuberculosis drug discovery and development. Transcriptome response to 1-hydroxy-5-R-pyridine-2(1H)-thiones, which were found to be highly active in vitro against actively growing and dormant nonculturable M. tuberculosis, revealed signs of copper toxicity. 1-Hydroxy-5-R-pyridine-2(1H)-thiones were found to form stable charged lipophilic complexes with Cu2+ ions that could transport into mycobacterial cells. Copper accumulated inside treated bacilli as subsequent metabolic destruction of the complex led to chemical transformation of 1-hydroxy-5-R-pyridine-2(1H)-thiones and release of free Cu2+ into the cytoplasm. 1-Hydroxy-5-R-pyridine-2(1H)-thiones are a potent class of Cu-dependent inhibitors of M. tuberculosis, and may control infection by impairment of copper homeostasis.

New 1-hydroxy-2-thiopyridine derivatives active against both replicating and dormant Mycobacterium tuberculosis.

Tuberculosis (TB) treatment is confounded by the range of metabolic states displayed by Mycobacterium tuberculosis, by the long duration required and by the increasing prevalence of drug-resistant strains. Latent TB infection is especially difficult to treat due to the phenotypic antibiotic resistance of non-replicating M. tuberculosis. Therefore, the development of new drugs effective against both active and latent TB infection is needed. New 1-hydroxy-2-thiopyridine derivatives were synthesized and found to be highly effective in vitro against both actively growing and dormant non-culturable M. tuberculosis. Such compounds are leads for the development of new drugs for all forms of TB including latent infection.